BER pathways play a major role in the repair of mutations caused by reactive oxygen species that are generated during aerobic metabolism, as described in Nature 362, 709-715(1993). Oxidative DNA damage has been implicated in the aetiology of degenerative diseases, ageing and cancer (Mutat. Res. 250, 3116 (1991), but evidence linking inherited deficiencies of BER to these diseases has, until recently, been lacking.
8-Oxo-7,8-dihydrodeoxyguanine (8-oxoG), the most stable product of oxidative DNA damage, is highly mutagenic, since it readily mispairs with A residues (Nature 349, 431-434 (1991)), leading to an increased frequency of spontaneous G:C→T:A transversion mutations in repair-deficient bacteria and yeast cells. In E. coli, three enzymes, mutM, mutY and mutT, function synergistically to protect cells from the deleterious effects of guanine oxidation (J Bacteriol. 174, 6321-6325(1992)). The mutM DNA glycosylase removes the oxidised base from 8-oxoG:C base pairs in duplex DNA; the mutY DNA glycosylase excises A residues misincorporated opposite unrepaired 8-oxoG during replication; and mutT is an 8-oxo-dGTPase preventing incorporation of 8-oxo-dGMP into nascent DNA. Human mutM, mutY and mutT homologues have been identified and termed hOGG1 (Proc. Natl. Acad. Sci.(USA) 94,8016-8020 (1997)), hMYH (or MYH) (J. Bactiol. 178, 3885-3892(1996)) and hMTH (J. Biol. Chem. 268,23524-23530 (1993)), respectively. Patent specification No. WO 97/33903 also discloses a human MutY polypeptide and DNA encoding it, together with its potential use in diagnosing a cancer or a susceptibility to a cancer.
Very recently in our International Patent Application WO (PCT/GB2002/003591) we have shown that mutations in the human base excision repair gene MYH (Accession No. NM—012222) cause an autosomal recessive trait characterised by multiple colorectal adenomas and high colorectal cancer risk.
Registers for patients and families with colorectal polyposis are widely established at regional or national level in many countries. They serve to co-ordinate proactive genetic testing, colonoscopic surveillance and surgical management across extended families. In the United Kingdom most polyposis registers are managed by regional clinical genetics services covering geographically defined populations of one to five million. The most important form of colorectal polyposis is familial adenomatous polyposis (FAP), an autosomal dominant disorder caused by mutations of the adenomatous polyposis coli (APC) gene. FAP is associated with hundreds or thousands of adenomatous polyps and leads to colorectal cancer in virtually all cases unless treated by prophylactic colectomy. Traditionally, all patients with >100 macroscopic colorectal adenomas are diagnosed as FAP. Classification of cases with less than 100 adenomas has been problematic. Some are associated with inherited mutations at specific locations within the APC gene and are classified as attenuated FAP (AFAP). The possible importance of other loci has been unclear.
The prevalence of MYH polyposis is unknown but it is unlikely to be confused with FAP or AFAP in families showing vertical transmission of polyposis. However, many patients with FAP/AFAP or with multiple colorectal adenomas (with or without colorectal cancer) occur as sporadic cases and others may have affected siblings with unaffected parents. Such cases may result from de novo APC gene mutations or gonadal mosaicism in a clinically unaffected parent. However, we hypothesised that some cases might be attributable to undiagnosed recessively transmitted MYH polyposis. If correct this would have important implications for family management. If an APC gene mutation is assumed, management for relatives of sporadic cases is based on a 1-in-2 risk to their offspring but a very low risk to their siblings. By contrast, the risks associated with MYH polyposis are 1-in-4 for the siblings of apparently sporadic cases, but extremely low for their offspring. Hence detection of MYH polyposis is important for accurate genetic counselling, genetic testing and effective planning of surveillance colonoscopy for extended families.
To identify families in which mutations of MYH rather than the APC gene might be causative, we applied the following selection criteria in six well established regional polyposis registers in the United Kingdom: 1) a family history showing no vertical transmission of polyposis 2) at least 10 colorectal adenomas with or without colorectal cancer in the index case 3) no clearly pathogenic mutation in the APC gene identified during genetic testing. We then sought MYH mutations in blood DNA samples from affected index cases. In previous reports (Al-Tassan et al. Nature Genet 2002, 30:227-232; Jones et al. Hum Mol Genet 2002, 11: 2961-7; Sieber et al. New Engl. J. Med. 2003, 348: 791-799), 31 out of 36 mutant alleles characterised in Caucasian patients with biallelic MYH mutations and colorectal polyposis were either Y165C (18 alleles) or G382D (13 alleles). Therefore, in Caucasian index cases we first assayed for these mutations by sequencing of exon 7 (for Y165C) and by BglII restriction enzyme digestion (for G382D). In cases heterozygous for either Y165C or G382D we screened for mutations affecting the second MYH allele by sequencing its 16 coding exons. Since different MYH mutations appear to be important in non-Caucasians (Jones et al. Hum Mol Genet 2002, 11: 2961-7) we sequenced all exons of MYH in all non-Caucasian index cases.
In addition to the mutations that we have previously identified in MYH (International Patent Application WO PCT/GB2002/003591), we identified four novel mutations: Q324X (C to T at nucleotide 970), W117R (T to A at nucleotide 349), 347−1 G to A and 891+3 A to C.